The misregulation of gene expression in ?-cells is a contributing factor to the pathogenesis of type 2 diabetes. However, gene expression is rarely studied in human ?-cells because of the lack of ?-cell lines and the relative difficulty in obtaining human Islets of Langerhans, leading to a reliance on rodent models of diabetes to answer fundamental questions relevant to human health. New analysis of the human genome has uncovered large numbers of regulatory DNA domains, many of which are human-specific and not conserved with other species. This suggests that aspects of ?-cell physiology and diabetes pathogenesis may be controlled by genes that are regulated in a human-specific fashion. Here we propose a model for a human- specific gene regulatory module in ?-cells between a microRNA, miR-375, and the transcriptional repressor CREM. MiR-375 is a negative regulator of insulin secretion, and CREM repressors are upregulated in rodent models of diabetes and have an adverse effect on insulin secretion. The purpose of this study is to test this model using both human and rat islets, and to compare the differences in gene expression between the two systems in response to cyclic adenosine monophosphate (cAMP) signaling. Cyclic-AMP potentiates glucose- stimulated insulin secretion and promotes long-term changes in ?-cell function, growth, and survival through regulation of gene expression. We propose that CREM (cAMP response element modulator) represses miR- 375 in a cAMP-dependent fashion in both humans and rodents, but that miR-375 in turn only binds to the human CREM messenger RNA (mRNA) leading to repression and a human-specific double-negative feedback loop. In Specific Aim 1, we will build on our preliminary evidence and test whether miR-375 is regulated by cAMP in human ?-cells, and whether CREM is modulating the cAMP-dependent transcriptional repression. We will study miR-375 expression using quantitative real-time PCR (qPCR), and we will use these tools in conjunction with CREM overexpression or knockdown to test whether CREM is necessary and sufficient for cAMP-dependent miR-375 repression. In Specific Aim 2, we will test whether miR-375 is binding specifically to the human CREM gene, and investigate the relationship between these two regulators using qPCR and a heterologous system. In Specific Aim 3, we will use insulin secretion assays and electrophysiology to study the effects of the miR-375?CREM feedback loop on ?-cell function. The work will be carried out in conjunction with Dr. David Jacobson at Vanderbilt University whose lab studies electrophysiology of pancreatic islets. Successful completion of this project will demonstrate that miR-375 and CREM regulate each other's expression in a double-negative feedback loop, specifically in human ?-cells. This model may have important consequences for the treatment of type 2 diabetes because CREM is activated by the cAMP-directed therapeutic exendin-4, and because miR-375 is reported to be overexpressed in diabetes. Misregulation of miR-375 may disrupt the normal functioning of this double-negative feedback loop in turn leading to misregulation of CREM. If this is the case, restoring the normal feedback loop through anti-miR-375 directed therapy may be warranted.